Since when nitric oxide (NO) was identified as a vasodilation factor, it has been found to be a physiologically active substance that plays an important role in regulating the biological functions. In addition to the above function, it was reported that NO has a platelet aggregation suppressing effect, a neurotransmitter releasing effect, and an effect to cause macrophage to exhibit antitumor and bactericidal activities (Moncada, S. and Higgs, A. (1993) N. Engl. J. Med. 329, 2002-2012).
NO is biologically synthesized by NO synthase (NOS) from L-arginine as a substrate. At present, three isozymes of this enzyme have been confirmed to exist (the brain type, the endothelial type, and the inducible type). Their chromosomal localization is also known (Knowles, R. G. and Moncada, S. (1994) Biochem. J. 298, 249-258).
Among these, the inducible type NOS (iNOS) can be expressed by applying endotoxins or cytokines to such cells as vascular smooth muscle cells (VSMC), hepatocytes, chondrocytes, or glial cells, to thereby induce gene transcription. Recently, mice deficient in this gene (knockout mice) have been developed. It was reported that the mice possess weaker defense capability against infections but exhibits alleviated symptoms of inflammation and sepsis as compared with the wild type (Wei, X. et al., (1995) Nature 375, 408-411; MacMicking, J. D. et al., (1995) Cell 81, 641-650). There is a report showing that iNOS is induced in any species by inflammatory conditions and that the suppression of its enzymatic activities and expression are effective for alleviating the inflammatory symptoms (Moncada, S. and Higgs, E. A. (1995) FASEB J. 9, 1319-1330). Further, in a sepsis model, the administration of iNOS enzyme inhibitors were found to be effective (Kerwin, J. F. et al., (1995) J. Med. Chem. 38, 4343-4362).
The endothelial type NOS (eNOS), on the other hand, was reported to participate in the homeostasis, especially in suppressing the rise in blood pressure, and is considered to play an important role in biological functions. Consequently, it has been desired to find a compound that does not affect the eNOS activity but specifically inhibits iNOS activity. However, since the primary structural domains of the proteins that inhibit the activities of these isozymes resemble each other very closely, none of the present NOS enzyme inhibitors are satisfactory in terms of their specificities.
On another aspect, it was confirmed that substances that generate NO can suppress vascular hypertrophy, thereby preventing arteriosclerosis and post-angioplasty vascular restenosis in animal models (Garg, U. C. and Hassid, A. J. (1989) J. Clin. Invest. 83, 1774-1777, Cooke, J. P. et al., (1992) J. Clin. Invest. 90, 1168-1172). It was also reported that the forced expression of the NOS gene in VSMC results in an increase of NO production accompanied by the suppression of hypertrophy of the inner membrane of VSMC (von der Leyen, H. E. et al., (1995) Proc. Natl. Acad. Sci. USA 92, 1137-1141). Therefore, it is expected that the generation of NO at the site of vascular hypertrophy can be effective in treating or preventing vascular hypertrophy.
The above-described facts suggest that, if iNOS isozyme-specific gene expression-regulating compounds are found out, the inhibitors will be useful as anti-inflammatory agents, while the inducers will be useful as agents which lead to therapeutic improvements in especially cardiovascular field.
Reporter genes are utilized for monitoring the expression of a certain gene simply and easily with high sensitivity, (Yokota, T., and Arai, K. (1993) Biomanual Series 4, Youdosha). Reporter genes are used instead of directly detecting the test gene expression and such genes that are widely being used at present include those whose expression products can be easily assayed, such as chloramphenicol acetyl transferase (CAT), .beta.-galactosidase (.beta.-Gal), and luciferase. The activity of the test DNA, whose regulatory activity is to be measured, can be detected with high sensitivity by inserting it into a plasmid at either upstream or downstream of the reporter gene.
The reporter genes used to examine the regulation of the mouse iNOS (miNOS) gene expression and the results were reported. For example, within the upstream region of the miNOS transcriptional start site (hereinafter referred to as "5'-flanking region"), there was found a region at about 1.7 kb upstream from the transcriptional start site, which is involved in the induction of the miNOS gene in response to lipopolysaccharides (LPS) or IFN-.gamma.. Furthermore, consensus sequence regions, to which transcription factors NF-.kappa.B and IRF-1 are considered to bind, were shown to be essential in the induction of the gene expression (Xie, Q. et al., (1993) J. Exp. Med. 117, 1779-1784, Vodovotz, Y. et al., (1993) J. Exp. Med. 178, 605-613, Martin, E. et al., (1994) J. Exp. Med. 180, 977-984, Xie, Q. et al., (1994) J. Biol. Chem. 269, 4705-4708). According to another report, the region was found within the 5'-flanking region of about 1.6 kb (Lowenstein, C. J. et al., (1993) Proc. Natl. Acad. Sci. USA 90, 9730-9734, Kamijo, R. et al., (1994) Science 263, 1612-1615). In both cases, however, the inducibility was less than 50-fold, which may not be strong enough to reflect the actual highly inducibility of the iNOS gene.
In contrast, it was difficult to prove the existence of cDNA of the human iNOS (hiNOS) gene. One of the reasons for this is that the cells, in which induction was effected by a single cytokine or a combination of two cytokines, were not discovered, unlike the case with the mouse macrophage or the rat VSMC. There is a report that hiNOS cannot be induced in the human macrophage even by a combination of three or more different kinds of cytokines (Weinberg, J. B. et al., (1995) Blood 86, 1184-1195). However, after the report of the first successful cloning of hiNOS cDNA by stimulating human hepatocytes with three or more different kinds of cytokines to induce the gene (Geller, D. A. et al., (1993) Proc. Natl. Acad. Sci. USA 90, 3491-3495), many reports of the hiNOS cDNA cloning emerged, but the regulatory mechanism of the induction has not been elucidated (Sherman, P. A. et al., (1993) Biochemistry 32, 11600-11605, Charles, I. G. et al., (1993) Proc. Natl. Acad. Sci. USA 90, 11419-11423, Hokari, A. et al., (1994) J. Biochem. 11, 575-581). At the same time, the complete structure of the hiNOS structural gene (FIG. 1) and its nucleotide sequence of the 5'-flanking region of approximately 0.4 kb were elucidated (Chartrain, N. A. et al., (1994) J. Biol. Chem. 269, 6765-6772).
On the other hand, the present inventors succeeded in cloning the iNOS gene from the rat vascular smooth muscle and reported to suggest that its cDNA is uniform throughout different tissues and species (Nunokawa, Y. et al., (1993) Biochem. Biophys. Res. Commun. 191, 89-94). Furthermore, the present inventors cloned a stretch of over 3.2 kb DNA, which is expected to contain the iNOS gene promoter region, determined the nucleotide sequence of the 5'-flanking region of approximately 1.5 kb, and reported that the sequence contains, as does the miNOS gene sequence, a consensus sequence thought to be regulated by interferon (IFN)-rand transcription factor NF-.kappa.B (Nunokawa, Y., et al., (1994) Biochem. Biophys. Res. Commun. 200, 802-807).
Thereafter, 16 kb of the 5'-flanking region of the hiNOS gene was studied using a reporter gene. The result showed that a region necessary for the induction of the hiNOS gene exists between 3.8 kb and 16 kb upstream from the transcriptional start site (de Vera, M. E. et al., (1996) Proc. Natl. Acad. Sci. USA 93, 1054-1059). However, the inducibility was still about 10-fold and the expression with no stimulation was quite strong. Thus, this report provides insufficient explanation for the actual and powerful induction of iNOS gene. In addition, the region up to 3.7 kb upstream from the transcriptional start site of the hiNOS gene was shown not to be involved in induction of the gene expression (Laubach, V. E. et al., (1994) Abstract Book of the 1st International Conference of Biochemistry and Molecular Biology of Nitric Oxide (UCLA Sunset Village, Los Angeles, Calif., USA), A16, Kleinert, H. et al., (1996) Mol. Pharmacol. 49, 15-21).
The present inventors generated cells transfected with a plasmid containing approximately 3.2 kb of the 5'-flanking region of the hiNOS gene, which had been previously cloned, linked to a reporter gene. The results indicated, contrary to those reported by using the miNOS promoter region, but similar to those reported by other groups using the hiNOS gene, that the reporter gene has been expressed even under non-inducing conditions when the 5'-flanking region of approximately 3.2 kb is used, and that the inducibility by cytokines is either absent or very weak (Nunokawa, Y. et al., (1996) Biochem. Biophys. Res. Commun. 223, 347-352). Moreover, the reporter gene expression was detected even in the cells transfected with the above plasmid that do not show the hiNOS induction by various cytokines (Nunokawa, Y. et al., (1996) Biochem. Biophys. Res. Commun. 223, 347-352). That is, it was found that the control of the hiNOS gene expression by inducers such as cytokines cannot be explained based solely on the promoter region which was inferred from the reported results with the miNOS gene. However, the reason for this has not been made clear at all so far.